The present invention relates to male infertility, and in particular to assays for determining fertility.
Infertility is diagnosed as the failure to become pregnant after one year of regular, unprotected intercourse. About ten percent of couples are infertile. Male factor infertility is the sole or contributing cause in about forty percent of these cases. In 1995, approximately 60,000 cycles of ART (Advanced Reproductive Technology) were performed in the United States to treat infertility. Of these procedures, approximately 90% involved in vitro fertilization at an average cost of $7,800.00 per cycle.
Semen analysis forms the basis of the initial evaluation for assessing male-factor. infertility. In general, two to three semen analyses are performed because semen quality normally fluctuates for a given individual. Subjects are normally encouraged to refrain from intercourse for 2 to 3 days prior to evaluation. Abstinence for a shorter time can decrease ejaculate volume, while prolonged abstinence can impair sperm motility. Traditional semen analysis evaluates a number of parameters, including, ejaculate volume, sperm count, sperm motility, forward progression, sperm morphology, pH, agglutination, leukospermia, and viscosity.
Sperm morphology is recognized as an important factor in semen analysis because it is a reflection of spermatogenic development. Traditionally, sperm have been classified according to the following morphologies: oval, amorphous, tapered, duplicated, and inrnature. However, determination of the percentage of normal spermatozoa with good morphology is highly subjective, and it is difficult to identify critical sperm morphological features that are responsible for fertility potential (Szczygiel and Kurpisz, Andrologia 31:63-75 [1999]). Additionally, in some instances, infertile men have sperm with apparently normal morphology. Furthermore, the results can be biased by damage incurred during normal preparation of the sperm for analysis (e.g., pipetting, centrifugation, and washing).
It is recognized that the identification of sperm abnormalities not apparent from semen analysis may lead to more appropriate and informed treatment plans for infertility (Szczygiel and Kurpisz, supra). However, there have been few recent efforts to develop methods that provide reliable prediction of fertility or fecundity based on sperm characteristics (Amann, J. Androl. 10(2):89-98 [1989]). Accordingly, what is needed in the art are objective semen quality assays that correlate to male factor infertility in the absence of morphological data suggesting otherwise, and that are unaffected by handling of the sample. It is desirable that such assays should be indicative of fertility.
The present invention relates to male infertility, and in particular to assays for determining fertility. In some embodiments of the present invention, methods for predicting fertility are provided. Accordingly, in some embodiments of the present invention, a method is provided for assaying fertility in an animal comprising a) providing a semen sample containing sperm; and b) measuring the amount of ubiquitin in the sample, wherein the amount of ubiquitin is indicative of fertility.
The present invention is not limited to a semen sample from a particular source. Indeed, it is contemplated that a variety of semen samples may be assayed. In some embodiments, the semen sample is obtained from a variety of animals, including, but not limited to, humans, cattle, sheep, pigs, horses, buffalo, bison and other domesticated and non-domesticated animals. In other embodiments, the sample is obtained by ejaculation, electroejaculation, or from the epididymis.
In other embodiments, the method of the present invention further comprises the steps of c) providing an antibody that binds to ubiquitin; and d) combining the semen sample with the antibody under conditions wherein the antibody binds to ubiquitihated sperm.
The present invention is not limited to any particular ubiquitin antibody. In some embodiments, the ubiquitin antibody is a polyclonal antibody, while in other embodiments, the ubiquitin antibody is a monoclonal antibody. In some particularly preferred embodiments, the antibody is selected from MAB 1510, AB 1690, Ubi-1, MK-11-3, MK-12-3, UCBA798/R5H, KM691, UG 9510, and U-5504.
The present invention is not limited to any particular technique for measuring ubiquitin or the degree of ubiquitination of sperm in the semen sample. Indeed, a variety of methods of determining the degree of ubiquitination are contemplated. In some embodiments, ubiquitination is assayed by immunocytochemical techniques wherein sperm that bind a labelled ubiquitin antibody are quantified. The present invention is also not limited to any particular method of quantitation. In some embodiments, the number of sperm within a given sample that are ubiquitinated is determined microscopically by counting the number of labelled sperm in at least one subsample of the semen sample. In other embodiments, the number of labelled sperm in a given sample is determined using a videoanalysis system in conjunction with fluorescence microscopy. In other embodiments, ubiquitination is assayed by immunocytochemical techniques, wherein sperm are sequentially exposed to a ubiquitin antibody and a labelled second antibody that binds to the ubiquitin antibody. In other embodiments of the present invention, flow cytometry is used to measure ubiquitin in a semen sample. In still further embodiments, the amount of ubiquitin in a semen sample is measured by enzyme-linked immunosorbant assay (ELISA).
The present invention is not limited to any particular labelled first or second antibodies. Indeed a variety of second antibodies are contemplated, including, but not limited to those labelled with fluorescent compounds (e.g., fluorescein, rhodamine), enzymatic markers (e.g., alkaline phosphatase, horseradish peroxidase), and colloidal gold.
In other embodiments, the present invention provides methods for assaying fertility in an animal. In some embodiments, the method comprises a) providing i) a test semen sample containing sperm; and ii) an antibody that binds to ubiquitin; b) combining the semen sample with the antibody under conditions wherein the antibody binds to ubiquitinated sperm; c) measuring the amount of ubiquitin in the sample; and, d) comparing the measured amount of ubiquitin in the sample with an amount of ubiquitin in a control sample from a donor of known fertility, wherein a greater amount of ubiquitination in the test semen sample as compared to the control sample is indicative of infertility.
In still other embodiments, the present invention provides kits for assaying sperm quality. In some embodiments, the kit comprises a) a first container containing an antibody that binds to ubiquitin; and b) a second container containing a control semen sample from a donor of known fertility. In further embodiments, the kit comprises an antibody that binds to the ubiquitin binding antibody. In still further embodiments, the kit comprises a labelled second antibody that binds to the antibody that binds ubiquitin. In other embodiments, the kit further comprises instructions for assaying fertility or sperm quality in an animal.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
As used herein, the term xe2x80x9cubiquitinxe2x80x9d refers to a relatively small protein (approximately 76 amino acid residues) found in all cells of higher organisms (See e.g., Ciechanover, Cell 79: 3-21 [1994], incorporated herein by reference) and other ubiquitin-like proteins sharing homology with ubiquitin. In preferred embodiments, ubiquitin and ubiquitin-like molecules will be recognized by the following antibodies: MAB 1510, AB 1690, Ubi-1, MK-11-3, MK-12-3, UCBA798/R5H, KM691, UG 9510, U-5504, P4D1 (Covance, Richmond, Calif.), 221M (Biomedia, Foster City, Calif.); 1 471 732 (Boehringer Mannheim, Indianapolis, Ind.), IB3 (Calbiochem, San Diego, Calif.); Z0458 (Dako Corp., Carpinteria, Calif.); NCL-UBIQ and NCL-UBIQm Novocastra (Novocastra, Newcastle upon Tyme, UK; Distributed in U.S. by Vector Labs Inc., Burlingame, Calif.), Ub(N-19), Ub(P1A), and Ub(C-20) (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.), Ub(P1A) Santa Cruz Biotechnology Inc., Santa Cruz, Calif.), and 1B4-UB (Serotech Inc., Raleigh, N.C.).
As used, herein, the term xe2x80x9cfertilityxe2x80x9d refers to the ability to conceive within one year of beginning unprotected intercourse.
As used herein, the term xe2x80x9cinfertilityxe2x80x9d refers to the inability to conceive after a year of unprotected intercourse.
As used herein, the term xe2x80x9csemen samplexe2x80x9d refers to any material containing sperm, whether processed or unprocessed, and includes ejaculates, electroejaculates, sperm isolated from testes or epididymes extended semen, sperm prepared by swim-up procedures, and sperm prepared by percoll gradient centrifugation.
As used herein, the term xe2x80x9cmeasuringxe2x80x9d refers to the act of determining the dimensions, quantity, or capacity of a material. When used in reference to ubiquitination of sperm in a sample, the term xe2x80x9cmeasuringxe2x80x9d encompasses determining the total amount of ubiquitin in a sample as well as determining the percentage or proportion of sperm that are ubiquitinated.
As used herein the term xe2x80x9cantibodyxe2x80x9d refers to a glycoprotein evoked in an animal by an immunogen (antigen). An antibody demonstrates specificity to the antigen, or, more specifically, to one or more epitopes contained in the immunogen. Native antibody comprises at least two light polypeptide chains and at least two.heavy polypeptide chains, including, but not limited to IgG, IgM, IgA, IgE, and IgD. Each of the heavy and light polypeptide chains contains at the amino terminal portion of the polypeptide chain a variable region (i.e., VH and VL. respectively), which, contains a binding domain that interacts with antigen. Each of the heavy and light polypeptide chains also comprises a constant region of the polypeptide chains (generally the carboxy terminal portion) which may mediate the binding of the immunoglobulin to host tissues or factors influencing various cells of the immune system, some phagocytic cells and the first component (C1q) of the classical complement system. The constant region of the light chains is referred to as the xe2x80x9cCL region,xe2x80x9d and the constant region of the heavy chain is referred to as the xe2x80x9cCH region.xe2x80x9d The constant region of the heavy chain comprises a CH1 region, a CH2 region, and a CH3 region. A portion of the heavy chain between the CH1 and CH2 regions is referred to as the hinge region (i.e., the xe2x80x9cH regionxe2x80x9d). The constant region of the heavy chain of the cell surface form of an antibody further comprises a spacer-transmembranal region (M1) and a cytoplasmic region (M2) of the membrane carboxy terminus. The secreted form of an antibody generally lacks the M1 and M2 regions.
As used herein, the term xe2x80x9cenzyme-linked immunosorbant assayxe2x80x9d refers to an immunoassay in which the amount of an antigen (e.g., ubiquitin) in a sample is quantitated by methods including, but not limited to, sandwich assays, competitive assays, and direct screening assays, and indirect assays (See e.g., Engvall, Methods in Enzymology, 70:419 [1980]; Scharpe et al., Clin. Chem. 22:733 [1976]; Schuurs et al., Immunoassay 1:229 (1980); Wisdom, Clin. Chem. 22:1243 [1976]).
As used herein, the term xe2x80x9cflow cytometryxe2x80x9d refers to an assay in which the proportion of a material (e.g., ubiquitinated sperm) in a sample is determined by labelling the material (e.g., by binding a labelled antibody to the material), causing a fluid stream containing the material to pass through a beam of light, separating the light emitted from the sample into constituent wavelengths by a series of filters and mirrors, and detecting the light.
As used herein, the term xe2x80x9cquantitatingxe2x80x9d refers to the act of determining the amount or proportion of a substance (e.g., ubiquitin or ubiquitinated sperm) in a sample.
As used herein, the term xe2x80x9cubiquitinated spermxe2x80x9d refers to sperm that contain one or more ubiquitin molecules conjugated to one or more proteins of the sperm that are not generally ubiquitinated or display low levels of ubiquitination in a normal sperm (i.e., sperm of normal morphology and physiology).
As used herein, the term xe2x80x9csubsamplexe2x80x9d refers to a sample population taken from a larger sample population.
As used herein, the phrase xe2x80x9cindicative of fertilityxe2x80x9d refers to a trait that correlates to fertility or infertility.
As used herein, the phrase xe2x80x9ccontrol sample from a donor of known fertilityxe2x80x9d refers to a semen sample that has been previously characterized as possessing an amount of ubiquitin that is consistent with known fertility. The control sample can be characterized by a number of methods, including those described herein and U.S. Pat. Nos. 5,962,241; 5,753,231; and 4,683,213, each of which is incorporated herein by reference. In one aspect, semen samples from known fertile.donors are within the definition. In another aspect, the control sample exhibits less ubiquitination than samples from infertile donors as measured by various quantitative techniques (e.g., ELISA, immunocytochemistry, or flow cytometry).
As used herein, the term xe2x80x9clabelled second or secondary antibodyxe2x80x9d refers to an antibody that is conjugated to a detectable group (e.g., fluorochrome, enzyme, colloidal gold) or otherwise includes a detectable group (e.g., a radioisotope) and is capable of binding to another antibody. In preferred embodiments, the labelled secondary antibody binds to a primary antibody that is bound to an antigen of interest (e.g., ubiquitin).
The present invention relates to male infertility, and in particular to assays for determining fertility. Sexual reproduction by fertilization requires normal structure and flawless functioning of both male and female gametes. This is assured at multiple levels, including not only the selection of the fittest sperm at the egg vitellus during fertilization, but also the preselection of both sperm and eggs prior to their release from gonads. In mammals, female gametes, oocytes, undergo dramatic reduction in number, during which only a few oocytes from the initial pool are allowed to grow into full size and become ovulated. Meanwhile, the vast majority of oocytes along with their somatic entourage of ovarian follicular cells undergo atresia (e.g., an apoptotic process), encompassing a relatively well characterized cascade of cellular events. In contrast with our knowledge of oocyte selection, there are only a handful of reports implicating a role for apoptosis in the preselection of male gametes, spermatozoa. For example, components of an active apoptotic pathway were found in both the spermatogenic cell lines (reviewed by Hikim and Swerdloff, Rev. Reprod. 4(1):38-47 [1999]) and in the mature sperm of mice (Weil et al., J. Cell Sci. 111:2707-2715 [1998]; Yin et al., Dev. Biol. 204:165-171 [1998] and men (Sakkas et al., Exp. Cell Res. 251:350-355 [1999]), but a definitive mechanism for sperm quality control is yet to be established.
Following the exit from the testis via testicular rete, the mammalian spermatozoa undergo maturation and storage in the epididymis. The mammalian epididymis is composed of three distinct compartments, namely the caput, corpus, and cauda; each of which has a specific role in sperm maturation, sustenance, transport, and storage. A number of proteins secreted in apocrine fashion by epididymal epithelium, have been implicated in sperm immobilization, stabilization of sperm perinuclear structure and acquisition of fertilizing potential (Kirchhoff, Rev. Reprod. 3:86-95 [1998]). This important function of the epididymis protects sperm from oxidative damage during storage and after release into female genital tract. It has also been reported that the droplets of residual cytoplasm carried over from testis (Hermo et al., Am. J. Anat. 183:107-124 [1988]), and most of the abnormal spermatozoa (Ramamohana et al., Theriogenology 14:1-12 [1980]; Roussel et al., Fertil. Steril. 18:509-516 [1967]) are resorbed during sperm descent down the epididymis.
Currently, in the bovine artificial insemination industry, bulls are evaluated for fertility by a process that takes from five to six years to complete. When a breeder examines a one-year-old, sexually mature bull, the breeder""s only source of information about the bull""s fertility is the pedigree information available on the animal. Testicular size and other gross physical characteristics of the animal provide little or no useful information relating to fertility. Typically such a bull first is bred to cows until as many as 200 offspring are produced and monitored for milk production or some other quantitative trait. In the case of dairy cattle, it takes up to four years to do this, because the daughters themselves must become sexually mature so that they can be impregnated, calve, and begin to produce milk. If daughter milk production is good, the bull is kept and included in the breeder""s general breeding program. Only at that time has the bull been bred to a population to a sufficiently large number of cows for the breeder to judge its fertility.
If a bull""s fertility is found to be unacceptably low, the bull is culled. Typically, only one out of seven bulls are kept after this lengthy evaluation evaluation of the bull""s progeny and fertility. In the meantime, the breeder has invested a large amount of money, time, and other resources to maintain and breed the bulls that are ultimately eliminated. Thus, detection and identification of bulls with low fertility at an early stage in the process provides considerable savings in time, money, and other resources.
It is not intended that the present invention be limited to particular mechanism of action. Indeed, an understanding of the mechanism is not necessary to make and use the present invention. However, the present inventors have discovered that the abnormal spermatozoa found in fertile males of several mammalian species including cattle and humans, are coated with ubiquitin or ubiquitin-like protein, a universal proteolytic marker, during epididymal passage. Furthermore, the degree of ubiquitination is correlated to fertility. Whereas a certain portion of the ubiquitinated defective sperm is resorbed before reaching the storage site in the cauda epididymis, other sperm are ejaculated and can be isolated from immotile sperm fraction. Therefore it appears that the ubiquitination of defective sperm may facilitate both their resorption prior to storage and immobilization prior to ejaculation. Thus, these mechanisms help prevent defective sperm from competing for an egg. These data are supported by the results of in situ investigations as well as by the reconstitution of this new epididymal function in vitro in the cultured epididymal epithelium. The finding of sperm ubiquitination in epididymis and its relationship to reproductive performance opens new possibilities for the diagnostics of male infertility, the evaluation of fertility, and may also offer new targets for contraceptives.
The following Description of the Invention is divided into the following topics: 1) Sources of Sperm for Analysis; 2) Antibodies Useful for Detection of Ubiquitin; 3) Methods for Quantifying Ubiquitination of Sperm; and 4) Fertility Assays.
1. Sources of Sperm for Analysis
The present invention provides methods for assaying fertility by determining the amount of ubiquitin in a semen sample (i.e., the degree of sperm ubiquitination). The method finds use in the analysis of semen samples from a variety of species (e.g., humans, bovines, primates, sheep, pigs, horses, rodents, camels, goats, bison, buffalo, llamas, foxes and ferrets). Furthermore, the samples may be collected by a variety of methods. In some embodiments of the present invention, the semen sample is from an ejaculate. In other embodiments, the semen sample is obtained by electroejaculation. In still other embodiments, the semen sample is obtained surgically from the epididymis. In some embodiments, the semen sample is analyzed without further processing except for preparation for flow cytometry, immunocytochemistry, or ELISA. However, in other embodiments, the sperm may be subjected to various preparation procedures known in the art (e.g., sperm swim-up or percoll gradient centrifugation).
2. Antibodies Useful for Detection of Ubiquitin
A variety of anti-ubiquitin antibodies are useful in the assays of the present invention. Examples of antibodies suitable for use in the present invention include, but are not limited to, MAB 1510 (Chemicon International, Inc. Temecula, Calif.); AB 1690 (Chemicon International, Inc. Temecula, Calif.); Ubi-1 (MAB1510) (Zymed Laboratories Inc., South San Francisco, Calif.); MK-11-3 (MBL Co, Ltd, Nagoya, Japan); MK-12-3 (MBL Co, Ltd, Nagoya, Japan); UCBA798/R5H (Accurate Chemical and Scientific Corp., Westbury, N.Y.); KM691 (Kamyia Biomedical Company, Seattle, Wash.); UG 9510 (Affiniti Research Products Ltd, Mamhead, UK); and U-5504 (Sigma, St. Louis, Mo.).
Alternatively, anti-ubiquitin antibodies are prepared as is known in the art using commercially available purified ubiquitin (Research Diagnostics, Inc., Flanders N.J.; See e.g., Ann. Rev. Biochem. 65:801 [1996]; J. Biol. Chem. 266:16476 [1990]; human ubiquitin 701-UB and 703-UB (RandD Systems, Minneapolis, Minn.); bovine ubiquitin U-6253, Sigma, St. Louis, Mo., Wilkinson et al., J. Biol. Chem., 256:9235 [1981]) to stimulate antibody production. The purified ubiquitin antigen finds use for the preparation of polyclonal, monoclonal, humanized, single chain and chimeric antibodies for use in the assays of the present invention. Thus, it is not intended that the present invention be limited to any particular type or class of antibody.
Various procedures known in the art may be used for the production of polyclonal antibodies to ubiquitin. For the production of antibody, various host animals can be immunized by injection with purified ubiquitin including but not limited to rabbits, mice, rats, sheep, goats, etc. In a preferred embodiment, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH)). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund""s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. 
For preparation of monoclonal antibodies directed toward ubiquitin, any technique that provides for the production of antibody molecules by continuous cell lines in culture as known in the art may be used (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). These include but are not limited to the hybridoma technique originally developed by Kxc3x6hler and Milstein (Kxc3x6hler and Milstein, Nature 256:495-497 [1975]), as well as the trioma technique, the human B-cell hybridoma technique (See e.g., Kozbor et al. Immunol. Today 4:72 [1983]), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 [1985]).
In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (See e.g., PCT/US90/02545). According to the invention, human antibodies may be used and can be obtained by using any method known in the art, including, but not limited to human hybridomas (Cote et al., Proc. Natl. Acad. Sci. U.S.A.80:2026-2030 [1983]) or by transforming human B cells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96 [1985]).
According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated by reference) can be adapted to produce ubiquitin-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science 246: 1275-1281 [1989]) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for ubiquitin.
Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(abxe2x80x2)2 fragment that can be produced by pepsin digestion of the antibody molecule; the Fabxe2x80x2 fragments that can be generated by reducing the disulfide bridges of the F(abxe2x80x2)2 fragment, and the Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent.
In some embodiments of the present invention, antibody binding is detected by detecting a label on the primary antibody. A variety of labels can be utilized depending on the nature of the assay. In some embodiments, the ubiquitin antibody is labelled with a fluorescent tag (e.g., fluorescein isothiocyanate, BODIPY, lucifer yellow, rhodamine isothiocyanate, texas red (sulfonyl chloride), lissamine rhodamine B, Cy3, Cy5, Cy7, allophycocyanin, cascade blue, succinimidyl esters of hydroxycoumarin, aminocoumarin, methoxycoumarin; these labels and their excitation and emission wavelengths are summarized in Table 1). In other embodiments, the ubiquitin antibody is labelled with an enzyme (e.g., alkaline phosphatase, horseradish peroxidase). In still further embodiments, the ubiquitin antibody is labelled with a radioactive tag (e.g., 125I, 35S, 3H) or colloidal gold. In other embodiments, the ubiquitin antibody is conjugated to biotin or strepavidin.
In other embodiments, the primary antibody is detected by detecting binding of a secondary antibody that recognizes the primary antibody (e.g., anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2, anti-rat IgG3, anti-rat IgG, anti-rat IgG1, anti-rat IgG2, anti-rat IgG3, anti-bovine IgM, anti-guinea pig IgG, and anti-sheep IgG; available from, e.g., Sigma, St. Louis Mo., and Gibco-BRL, Gaithersburg, Md.) or reagent to the primary antibody (e.g., fluorescein labelled biotin, rhodamine labelled biotin, fluorescein labelled strepavidin, rhodamine labelled strepavidin). In a further embodiment, the secondary antibody is labelled (e.g., fluorescent, radioactive, or enzyme labels as described above for the ubiquitin primary antibody).
In still other embodiments, ubiquitin is detected by binding to a binding molecule other than an antibody. For example, RNA and DNA molecules that bind to ubiquitin can be identified by the SELEX procedure. The basic SELEX procedure is described in U.S. Pat. Nos. 5,475,096; 5,270,163; and 5,475,096; and in PCT publications WO 97/38134, WO 98/33941 and WO 99/07724, all of which are herein incorporated by reference. The SELEX procedure allows identification of a nucleic acid molecules with unique sequences, each of which has the property of binding specifically to a desired target compound or molecule. In some particularly preferred embodiments, the binding molecule is labelled as described above.
3. Methods for Quantifying Ubiquitination of Sperm
The foregoing antibodies can be used in methods known in the art relating to the localization and presence of ubiquitin, and measuring levels thereof in appropriate biological samples (e.g., semen and sperm samples). The biological samples can then be tested directly for the presence of ubiquitin using an appropriate strategy (e.g., flow cytometry, immunocytochemistry, ELISA or radioirnmunoassay) and format (e.g., microwells, dipstick, etc; See e.g., International Patent Publication WO 93/03367). Alternatively, proteins in the sample can be size separated (e.g., by polyacrylamide gel electrophoresis (PAGE), in the presence or absence of sodium dodecyl sulfate (SDS). In these size separation methods the presence of ubiquitin can be detected by methods such as immunoblotting (e.g., Western blotting)).
In some preferred embodiments of the present invention, the number of ubiquitinated sperm in a semen sample is determined by immunocytochemical techniques known in the art. In some embodiments, ubiquitinated sperm are detected in a semen sample by fixing sperm from the semen sample to a microscope slide (or in the case of electron microscopy pelleting the sample by centrifugation), labelling the sperm with a labelled (e.g., fluorescently, enzymatically, or radioactively, or colloidal gold tagged) ubiquitin antibody, and detecting the presence of the tag (e.g., by fluorescent microscopy, light microscopy, autoradiography, or electron microscopy). In other embodiments, the sperm are first bound to an unlabelled ubiquitin antibody (i.e., a primary antibody), and then the ubiquitin antibody is bound to a tagged. (e.g., fluorescently, enzymatically, or radioactively labelled) secondary antibody. A number of immunocytochemical strategies and techniques find use in the present invention. The present immunocytochemical assays can be conducted in a direct manner (e.g., the ubiquitin antibody is labelled) or indirect manner (e.g., a labelled second antibody is used to detect bound ubiquitin antibody). In some embodiments, the second antibody is conjugated to biotin or avidin so that it can be detected by a biotinylated or avidin conjugated tag. In still other embodiments, methods known in the art such as PAP (peroxidase-antiperoxidase) or APAAP (alkaline phosphatase-antialkaline phosphatase) are used to detect ubiquitin. In addition, antigen retrieval methods such as enzyme digestion and HMAR (heat mediated antigen retrieval techniques such as xe2x80x9cmicrowavingxe2x80x9d and xe2x80x9cpressure cookingxe2x80x9d) find use in the present invention.
In some particularly preferred embodiments, the primary or secondary antibody is fluorescently tagged (e.g., with fluorescein or rhodamine) and ubiquitinated sperm are quantified by fluorescence microscopy. In some embodiments, the microscope slide is divided into a grid, the total number of sperm within the grid (e.g., a subsample) is determined via light microscopy or via fluorescent microscopy if the sperm are labelled with a DNA stain (e.g., DAPI, Molecular Probes, Eugene, Oreg.) or cytoskeleton specific stain (e.g., rhodamine-phalloidin, Molecular Probes, Eugene, Oreg.), and the number of ubiquitinated sperm observed by exciting the fluorescently tagged antibody. In other embodiments, the subsample is simply the number of sperm within a viewing field of the microscope. The percentage of ubiquitinated sperm is determined by dividing the number of sperm tagged with the labelled antibody by the total number of sperm. In some embodiments, this process is repeated for a number of subsamples (e.g., 3, 4, 5 or more subsamples) on a given slide. In other embodiments, the process is repeated for several (e.g., 2 or more) ejaculates or semen samples from an individual male. In some particularly preferred embodiments, a video image analysis system (e.g., Image 1) is utilized to count the number of sperm exhibiting a threshold level of fluorescence. In some embodiments, the test semen samples are prepared in parallel with control semen samples from a donor of known fertility. An increased amount of ubiquitinated sperm in the test semen sample is indicative of a decreased level of fertility, while approximately equal or lower levels of ubiquitination as compared to the control sample are indicative of good fertility.
In other preferred embodiments of the present invention, the level of ubiquitination in a semen sample is determined by ELISA. The present invention contemplates the use of a number of different types of ELISA formats to analyze ubiquitination in semen samples, including, but not limited to, sandwich assays, competitive assays, and direct screening assays (See e.g., Engvall, Methods in Enzymology, 70:419 [1980]; Scharpe et al., Clin. Chem. 22:733 [1976]; Schuurs et al., Immunoassay 1:229 (1980); Wisdom, Clin. Chem. 22:1243 [1976]).
In some preferred embodiments, a test semen sample is diluted (e.g., from about 3xc3x97107 sperm per ml to about 1xc3x97104 sperm per ml) and an aliquot (e.g., about 1 to 50 xcexcl) added to a well in ELISA assay plate (e.g., a Corning-Costar 96 well plate). The samples are washed and then a ubiquitin antibody is added and allowed to bind. In some embodiments, the ubiquitin antibody is tagged with an enzymatic label (e.g., alkaline phosphatase or horseradish peroxidase). In other embodiments, an enzymatically tagged second antibody is used to detect the bound ubiquitin antibody. In some particularly preferred embodiments, the second antibody is conjugated to alkaline phosphatase. In some embodiments, the presence of alkaline phosphatase is detected by adding alkaline phosphatase substrate (Zymed, So. San Francisco, Calif.) to the well, incubating, and reading the results at 405 nm in a photocolorimeter, plate reader, or spectrophotometer. In some embodiments, control semen samples from donors of known fertility are included and analyzed in parallel with the test semen samples. An increased amount of ubiquitinated sperm in the test semen sample as assayed by an increase in the signal utilized in the ELISA is indicative of a decreased fertility, while approximately equal or lower levels of ubiquitination as compared to the control sample are indicative of good fertility.
In still other preferred embodiments of the present invention, the level of ubiquitination in a semen sample is determined by flow cytometry. Flow cytometry generally involves the use of a fluid stream containing cells (e.g., sperm) that is passed through a beam of light, usually generated by a laser (e.g., argon, helium-neon, krypton, or dye laser), so that one cell at a time passes through the light path. The photons of light, which are scattered and emitted by the cells following their interaction with the laser beam, are separated into constituent wavelengths by a series of filters and mirrors. The separated light falls on a detector to generate an analog signal, this signal is then converted to a digital signal, which is accumulated and displayed in frequency distribution (i.e., histogram). The resulting value obtained is proportional to the amount of light emitted from each individual cell (See e.g., McCoy, Flow Cytometry and Clinical Diagnosis, Karen et al., eds., ASCP Press, Chicago, p. 26-55 [1994]; Flow Cytometry: A Practical Approach, Ormerod, ed., IRL Press, Oxford [1994]; Handbook of Flow Cytometry Methods, Robinson ed., Wiley-Liss, New York, 1993).
In some embodiments, the sperm from a semen sample are treated (e.g., with formaldehyde) and incubated with a primary ubiquitin antibody (e.g., KM 693 or MK-12-3). The sperm-primary antibody complexes are then incubated with fluorescently tagged second antibody (e.g., FITC conjugated goat anti-mouse IgM or IgG). The labelled sperm are then analyzed via flow cytometry. In some preferred embodiments, at least one control sample from a donor of known fertility is analyzed in parallel with test semen samples. In some embodiments, the data are analyzed by plotting the relative fluorescence for each sample and recording the median value. The median value provides the percentage of cells with average fluorescence and is increased proportionally to the increase in the number of labelled cells (i.e., the median value is the percent of sperm with average specific fluorescence). An increase in the median value of the test semen sample is correlated with an increase in ubiquitination and is indicative of a decreased level of fertility. In contrast, lower levels of ubiquitination (i.e., indicated by a lower median value) as compared to the control sample are indicative of good fertility.
In other embodiments of the present invention, fertility is assessed by combining ubiquitination analysis with traditional semen quality analysis parameters, including, but not limited to, ejaculate volume, sperm count, sperm motility, forward progression, sperm morphology, pH, agglutination, leukospermia, and viscosity. In infertile couples, the pregnancy history, fertilization, and cleavage rates after in vitro fertilization may be combined with some or all of the above parameters.
4. Fertility Assay Kits
In some embodiments of the present invention, kits are provided that contain reagents for performing the assays described above. In some embodiments, the kits include a container of ubiquitin antibody. In further embodiments, the kits include a container of semen sample from a donor of known fertility. In some embodiments where the primary antibody is not labelled, the kit further include a container of a labelled second antibody. In some preferred embodiments, the kit also includes directions for performing the ubiquitin assays described above and in the Experimental section below. The directions include methods for preparing the semen sample, fixing the sperm, binding the primary ubiquitin antibody to the sperm, labelling the primary antibody-sperm complex with a labelled second antibody, and detecting the complex via flow cytometry, ELISA, or immunocytochemistry. The kit instructions also provide methods for quantifying the results and comparing the results obtained for test samples to results obtained for the control samples, and for correlating these results with fertility.